1. Field of the Invention
The present invention relates generally to the field of virology and vaccine development. More specifically, the present invention provides a method to attenuate encephalitogenic alphaviruses including but not limited to VEEV, EEEV and WEEV. The attenuated phenotype is irreversible and thus, can be effective as human and/or veterinary vaccines in immunogenic composition(s).
2. Description of the Related Art
The Alphavirus genus in the Togaviridae family includes a number of important human and animal pathogens (15). Alphaviruses are currently classified into 6 antigenic complexes and are widely distributed both in the New and the Old World. They are efficiently transmitted by mosquitoes, in which they cause a persistent, lifelong infection that does not noticeably affect biological functions of the vectors. In vertebrates, the infection is acute and characterized by high-titer viremia, rash and fever and encephalitis until the death of the infected host or clearance of the virus by the immune system. The encephalitogenic alphaviruses, including Venezuelan (VEEV), eastern (EEEV) and western equine encephalitis (WEEV) viruses, represent a continuous public health threat in the U.S. (40, 47-49). They circulate in the Central, South and North Americas and have an ability to cause fatal disease in humans and horses. During VEEV epizootics, equine mortality can reach 83%, and, in humans, this virus produces a severe temporary immunodeficiency and a greatly debilitating and sometimes fatal disease (41). The overall mortality rate is below 1%, but the neurological disease, including disorientation, ataxia, mental depression, and convulsions, can be detected in up to 14% of all infected individuals, especially children (23). Sequelae of VEEV-related clinical encephalitis in humans are also described (10, 27).
The VEEV genome is represented by a single-stranded RNA molecule of positive polarity of almost 12-kb. It mimics the structure of cellular mRNA, in which it contains a Cap at the 5′ terminus and a poly(A) tail at the 3′ end of the RNA. VEEV genome has been cloned in a cDNA form (24) that allows a wide variety of genetic manipulation to be undertaken.
The current experimental vaccine against VEEV infection was developed four decades ago by serial passaging of the virulent, subtype IAB Trimidad Donkey (TRD) VEEV strain in guinea pig heart cell cultures (3). Presently, TC-83 is still the only available vaccine for laboratory workers and military personnel. Over 8,000 humans have been vaccinated during the past 4 decades (2, 6, 36), and the cumulative data unambiguously demonstrated that nearly 40% of vaccinated people develop a disease with some symptoms typical of natural VEEV infection, including a febrile, systemic illness and other adverse effects (2, 3, 21). No effective antivirals have been developed against this virus as well.
In spite of the continuous threat of VEEV epidemics, the biology of this virus has been studied less intensively than that of other, less pathogenic alphaviruses, such as Sindbis (SINV) and Semliki Forest (SFV) viruses. This situation can be partially explained by the fact that for a long time, it was believed that the latter viruses represent excellent models for studying the mechanism of alphavirus replication, virus-host interactions and encephalitis development (14). However, very strong differences in pathogenesis and the severity of the caused diseases suggest that this may not exactly be the case. Moreover, the results from recent comparative studies with the Old World alphaviruses (SINV and SFV) and the New World alphaviruses (VEEV and EEEV) (1, 9, 11-13, 35, 45) demonstrated that both of these groups have developed the ability to interfere with cellular transcription and use it as a means of downregulating cellular antiviral reactions. However, the mechanism of transcription inhibition appears to be fundamentally different, and while the Old World alphaviruses use nsP2 to inhibit cellular transcription (11), the more encephalitogenic VEEV and EEEV use their capsid protein for the same function (1, 12). Expression of the latter protein by different vectors is sufficient for induction of cell death and cytopathic effect (CPE) in tissue culture, and this effect strongly correlates with the inhibition of transcription of cellular messenger and ribosomal RNAs. Moreover, the replacement of structural genes in VEEV by those derived from SINV made the chimeric virus strongly less cytopathic and incapable of interfering with the development of an antiviral reaction developing in the cells having no defect in IFN-a/b induction and signaling (12).
Despite this, prior art is deficient in an immunogenic composition(s) that will prevent and treat infection caused by encephalitogenic alphavirus. The current invention fulfils this long standing need in the art.